Glucocorticoid antagonizing factor (GAF) is to be purified from serum or from the supernate of peritoneal macrophages cultured in the presence of lipopolysaccharide (LPS). Purification will be achieved through the use of chromatographic procedures. Antibody against purified GAF will be raised in goats or rabbits and used in a variety of ways designed to elucidate its role in normal and in LPS-poisoned animals. This will be accomplished by assessing the consequences of its neutralization in vivo, especially whether endotoxemic shock can be diminished or reversed by injecting anti-GAF immunoglobulin. The nature of the antagonism between GAF and glucocorticoids will be determined through its effect on the binding of radiolabeled dexamethasone to cytoplasmic receptor proteins in Reuber H35 hepatoma cells or liver parenchymal cells. The availability of anti-GAF antiserum will make if feasible to prepare and isolate in pure form by affinity chromatography radiolabeled GAF. Its distribution within cells can then be done by autoradiography. The site of action of GAF that enables it to block in hepatoma cells, liver and kidney the glucocorticoid induction of phosphoenolpyruvate carboxykinase (PEPCK) will be determined by isolating the mRNA for the enzyme (probably from kidney) and using a wheat germ cell-free protein synthesizing system to make radiolabeled PEPCK. Anti-enzyme antibody will be added to the reaction mixture to precipitate PEPCK. The necessary combination of control and experimental preparations will make it possible to distinguish a block at the transcriptional level from one at the translational level.